The overall objective is to understand the functional significance of selected modified nucleotides in mammalian tRNA, with emphasis on human tRNA. Our approach has been first to determine the primary structure of several normal mammalian tRNAs and then compare the functions of tRNAs which show alterations in modified nucleotide content. During this past year our research has been concentrated in three areas, elucidating the complete nucleotide sequence of normal and tumor tRNA Asn, determining the function of ribothymidine and Q nucleosides in mammalian tRNA, and characterising human mitochondrial tRNAs and their genes. Ribothymidine alters the overall structure of selected mammalian tRNAs, such that they function at an optimum rate in the translocation step in protein synthesis, since we observed that the differing ribothymidine content of human, beef, rat and rabbit tRNA Phe did not affect ribosome binding but did alter the maximum velocity of protein synthesis. These studies will now be extended to a detailed investigation of the effect of ribothymidine content of tRNA Phe on the translocation step of mammalian protein synthesis. In the studies on Q nucleoside, we will now compare the ability of the normal Q-containing and tumor G-containing tRNA Asn in codon binding, aminoacylation, in vitro protein synthesis and in priming for AMV-reverse transcriptase. We also anticipate completing the nucleotide sequence of several mammalian mitochondrial tRNAs and using the tRNA genes to investigate the biosynthesis of mitochondrial tRNAs and their modified nucleotides.